Fertilization occurs after completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. In mammals, secretion is triggered by ZP3, a glycoprotein component of the egg's extracellular matrix, or zona pellucida (ZP). The long range goal of this project is to understand the mechanism of ZP3 signal transduction during the acrosome reaction. We have previously shown that transient receptor potential canonical (TRPC) channels play an essential role in ZP3-induced acrosome reaction in mouse sperm. This research proposal focuses on TRPC-effector proteins that participate in events downstream of ZP3 signal initiation. Using a yeast-two hybrid we have identified a novel sperm protein, enkurin, which binds to both TRPC channels and phosphatidylinositol-3-phosphate (PI3K). The demonstration of an enkurin- PI3K interaction prompted us to investigate a role for PI3K in the acrosome reaction. We present preliminary data showing that ZP3 stimulates the production of the PI3K product phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 is required to drive the acrosome reaction. There are two specific aims: 1) A male germline enkurin null mouse will be generated to determine the function of enkurin function in sperm;and 2) Identify downstream effectors of PI3K during the ZP-induced AR in mouse sperm;preliminary data suggests that AKT is involved in the ZP-acrosome reaction signaling pathway in mouse sperm. The next step is to identify the substrates of this kinase during acrosomal exocytosis. PROJECT NARRATIVE: Health problems are posed when fertilization is either unbounded or impeded. In this regard, the acrosome reaction is an important regulatory event during mammalian gamete interaction, with unregulated exocytosis associated with failure of fertilization. These studies define downstream effector proteins that are essential components of the ZP3 signaling pathway and may provide new targets for the control of fertilization.